Two separate effects contribute to regulatory T cell defect in systemic lupus erythematosus patients and their unaffected relatives

Forkhead box P3 (FoxP3)⁺ regulatory T cells (T_regs) are functionally deficient in systemic lupus erythematosus (SLE), characterized by reduced surface CD25 [the interleukin (IL)‐2 receptor alpha chain]. Low‐dose IL‐2 therapy is a promising current approach to correct this defect. To elucidate the origins of the SLE T_reg phenotype, we studied its role through developmentally defined regulatory T cell (T_reg) subsets in 45 SLE patients, 103 SLE‐unaffected first‐degree relatives and 61 unrelated healthy control subjects, and genetic association with the CD25‐encoding IL2RA locus. We identified two separate, uncorrelated effects contributing to T_reg CD25. (1) SLE patients and unaffected relatives remarkably shared CD25 reduction versus controls, particularly in the developmentally earliest CD4⁺FoxP3⁺CD45RO^–CD31⁺ recent thymic emigrant T_regs. This first component effect influenced the proportions of circulating CD4⁺FoxP3^highCD45RO⁺ activated T_regs. (2) In contrast, patients and unaffected relatives differed sharply in their activated T_reg CD25 state: while relatives as control subjects up‐regulated CD25 strongly in these cells during differentiation from naive T_regs, SLE patients specifically failed to do so. This CD25 up‐regulation depended upon IL2RA genetic variation and was related functionally to the proliferation of activated T_regs, but not to their circulating numbers. Both effects were found related to T cell IL‐2 production. Our results point to (1) a heritable, intrathymic mechanism responsible for reduced CD25 on early T_regs and decreased activation capacity in an extended risk population, which can be compensated by (2) functionally independent CD25 up‐regulation upon peripheral T_reg activation that is selectively deficient in patients. We expect that T_reg‐directed therapies can be monitored more effectively when taking this distinction into account.     

CD4+CD45RA−FoxP3high activated regulatory T cells are functionally impaired and related to residual insulin‐secreting capacity in patients with type 1 diabetes

Accumulating lines of evidence have suggested that regulatory T cells (T_regs) play a central role in T cell-mediated immune response and the development of type 1A and fulminant type 1 diabetes. CD4⁺forkhead box protein 3 (FoxP3)⁺ T cells are composed of three phenotypically and functionally distinct subpopulations; CD45RA⁺FoxP3^low resting T_regs (r-T_regs), CD45RA⁻FoxP3^high activated T_regs (a-T_regs) and CD45RA⁻FoxP3^low non-suppressive T cells (non-T_regs). We aimed to clarify the frequency of these three subpopulations in CD4⁺FoxP3⁺ T cells and the function of a-T_regs with reference to subtypes of type 1 diabetes. We examined 20 patients with type 1A diabetes, 15 patients with fulminant type 1 diabetes, 20 patients with type 2 diabetes and 30 healthy control subjects. A flow cytometric analysis in the peripheral blood was performed for the frequency analysis. The suppressive function of a-T_regs was assessed by their ability to suppress the proliferation of responder cells in a 1/2:1 co-culture. A flow cytometric analysis in the peripheral blood demonstrated that the frequency of a-T_regs was significantly higher in type 1A diabetes, but not in fulminant type 1 diabetes, than the controls. Further, the proportion of a-T_regs among CD4⁺FoxP3⁺ T cells was significantly higher in patients with type 1A diabetes with detectable C-peptide but not in patients with type 1A diabetes without it and with fulminant type 1 diabetes. A proliferation suppression assay showed that a-T_regs were functionally impaired both in fulminant type 1 diabetes and in type 1A diabetes. In conclusion, a-T_regs were functionally impaired, related to residual insulin-secreting capacity and may be associated with the development of type 1 diabetes.     

Regulatory T‐cells in acute dengue viral infection

Although regulatory T‐cells (T_regs) have been shown to be expanded in acute dengue, their role in pathogenesis and their relationship to clinical disease severity and extent of viraemia have not been fully evaluated. The frequency of T_regs was assessed in 56 adult patients with acute dengue by determining the proportion of forkhead box protein 3 (FoxP3) expressing CD4⁺ CD25⁺T‐cells (FoxP3⁺ cells). Dengue virus (DENV) viral loads were measured by quantitative real‐time polymerase chain reaction (PCR) and DENV‐specific T‐cell responses were measured by ex‐vivo interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assays to overlapping peptide pools of DENV‐NS3, NS1 and NS5. CD45RA and CCR4 were used to phenotype different subsets of T‐cells and their suppressive potential was assessed by their expression of cytotoxic T lymphocyte‐antigen 4 (CTLA‐4) and Fas. While the frequency of FoxP3⁺ cells in patients was significantly higher (P < 0·0001) when compared to healthy individuals, they did not show any relationship with clinical disease severity or the degree of viraemia. The frequency of FoxP3⁺ cells did not correlate with either ex‐vivo IFN‐γ DENV‐NS3‐, NS5‐ or NS1‐specific T‐cell responses. FoxP3⁺ cells of patients with acute dengue were predominantly CD45RA⁺ FoxP3^low, followed by CD45RA‐FoxP3^low, with only a small proportion of FoxP3⁺ cells being of the highly suppressive effector Treg subtype. Expression of CCR4 was also low in the majority of T‐cells, with only CCR4 only being expressed at high levels in the effector T_reg population. Therefore, although FoxP3⁺ cells are expanded in acute dengue, they predominantly consist of naive T_regs, with poor suppressive capacity.     

The role of regulatory T cell (Treg) subsets in gestational diabetes mellitus

Physiological changes during normal pregnancy are characterized by an inflammatory immune response and insulin resistance. Therefore, we hypothesize that gestational diabetes mellitus (GDM) may be caused by an inappropriate adaption of the maternal immune system to pregnancy. In this study we examined the role of regulatory T cell (T_reg) differentiation for the development of GDM during pregnancy. We used six-colour flow cytometric analysis to demonstrate that the total CD4⁺CD127^low+/−CD25⁺ forkhead box protein 3 (FoxP3⁺) T_reg pool consists of four different T_reg subsets: naive CD45RA⁺ T_regs, HLA-DR⁻CD45RA⁻ memory T_regs (DR⁻ T_regs) and the highly differentiated and activated HLA-DR^low+CD45RA⁻ and HLA-DR^high+CD45RA⁻ memory T_regs (DR^low+ and DR^high+ T_regs). Compared to healthy pregnancies, the percentage of CD4⁺CD127^low+/−CD25⁺FoxP3⁺ T_regs within the total CD4⁺ T helper cell pool was not different in patients affected by GDM. However, the suppressive activity of the total CD4⁺CD127^low+/−CD25⁺ T_reg pool was significantly reduced in GDM patients. The composition of the total T_reg pool changed in the way that its percentage of naive CD45RA⁺ T_regs was decreased significantly in both patients with dietary-adjusted GDM and patients with insulin-dependent GDM. In contrast, the percentage of DR⁻-memory T_regs was increased significantly in patients with dietary-adjusted GDM, while the percentage of DR^low+ and DR^high+ memory T_regs was increased significantly in patients with insulin-dependent GDM. Hence, our findings propose that alterations in homeostatic parameters related to the development and function of naive and memory T_regs may cause the reduction of the suppressive capacity of the total T_reg pool in GDM patients. However, as this is an exploratory analysis, the results are only suggestive and require further validation.     

Increased levels of regulatory T cells (Tregs) in human immunodeficiency virus‐infected patients after 5 years of highly active anti‐retroviral therapy may be due to increased thymic production of naive Tregs

This study determines levels of regulatory T cells (T_regs), naive T_regs, immune activation and cytokine patterns in 15 adult human immunodeficiency virus (HIV)‐infected patients receiving prolonged highly active anti‐retroviral therapy (HAART) who have known thymic output, and explores if naive T_regs may represent recent thymic emigrant T_regs. HIV‐infected patients treated with HAART with a median of 1 and 5 years were compared with healthy controls. Percentages of T_regs (CD3⁺CD4⁺CD25⁺CD127^low), naive T_regs (CD3⁺CD4⁺CD25⁺CD45RA⁺) and activation markers (CD38⁺human leucocyte antigen D‐related) were determined by flow cytometry. Forkhead box P3 mRNA expression and T cell receptor excision circles (T_REC) content in CD4⁺ cells were determined by polymerase chain reaction and cytokines analysed with Luminex technology. Levels of T_regs were significantly higher in HIV‐infected patients compared with controls, both after 1 and 5 years of HAART (P < 0·001), despite fully suppressed HIV‐RNA and normalization of both CD4 counts, immune activation and cytokine patterns. Furthermore, levels of naive T_regs were elevated significantly in HIV‐infected patients (P < 0·001) and were associated with thymic output measured as the T_REC frequency in CD4⁺ cells (P = 0·038). In summary, T_reg levels in HIV‐infected patients are elevated even after 5 years of HAART. Increased thymic production of naive T_regs may contribute to higher T_reg levels in HIV‐infection.     

Level,phenotype and activation status of CD4+FoxP3+ regulatory T cells in patients chronically infected with human immunodeficiency virus and/or hepatitis C virus

CD4⁺ regulatory T (T_reg) cells have been involved in impaired immunity and persistence of viral infections. Herein, we report the level, phenotype and activation status of T_reg cells in patients chronically infected with human immunodeficiency virus (HIV) and/or hepatitis C virus (HCV). Expression of CD25, CD45RA, CD27, CD127 and CD38 was assessed on these cells using polychromatic flow cytometry in 20 healthy controls, 20 HIV‐monoinfected, 20 HCV‐monoinfected and 31 HIV/HCV‐co‐infected patients. T_reg cells were defined as CD4⁺forkhead box P3 (FoxP3)⁺. The percentage of T_reg cells was increased significantly in HIV patients compared with controls. Moreover, there was a significant inverse correlation between CD4 counts and T_reg cell levels. Fewer than 50% of T_reg cells expressed CD25, with differences in terms of CD127 expression between CD25⁺ and CD25(^–) T_reg cells. CD4⁺Foxp3⁺ T_reg cells displayed predominantly a central memory phenotype (CD45RA^–CD27⁺), without differences between patients and healthy controls. Activated T_reg cells were increased in HIV patients, particularly considering the central memory subset. In summary, HIV infection, but not HCV, induces an up‐regulation of highly activated T_reg cells, which increases in parallel with CD4 depletion. Hypothetically, this might contribute to the accelerated course of HCV‐related liver disease in HIV‐immunosuppressed patients.     

Obtaining regulatory T cells from uraemic patients awaiting kidney transplantation for use in clinical trials

Adoptive transfer of regulatory T cells (T_regs) has been proposed for use as a cellular therapy to induce transplantation tolerance. Preclinical data are encouraging, and clinical trials with T_reg therapy are anticipated. In this study, we investigate different strategies for the isolation and expansion of CD4⁺CD25^highCD127^low T_regs from uraemic patients. We use allogeneic dendritic cells (DCs) as feeder cells for the expansion and compare T_reg preparations isolated by either fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) that have been expanded subsequently with either mature or tolerogenic DCs. Expanded T_reg preparations have been characterized by their purity, cytokine production and in‐vitro suppressive ability. The results show that T_reg preparations can be isolated from uraemic patients by both FACS and MACS. Also, the type of feeder cells used in the expansion affects both the purity and the functional properties of the T_reg preparations. In particular, FACS‐sorted T_reg preparations expanded with mature DCs secrete more interleukin (IL)‐10 and granzyme B than FACS‐sorted T_reg preparations expanded with tolerogenic DCs. This is a direct comparison between different isolation techniques and expansion protocols with T_regs from uraemic patients that may guide future efforts to produce clinical‐grade T_regs for use in kidney transplantation.     

Differentiation of ICOS+ and ICOS− recent thymic emigrant regulatory T cells (RTE Tregs) during normal pregnancy,pre‐eclampsia and HELLP syndrome

Two different subsets of naturally occurring regulatory T cells (nT_regs), defined by their expression of the inducible co‐stimulatory (ICOS) molecule, are produced by the human thymus. To examine the differentiation of ICOS⁺ and ICOS^?CD45RA⁺CD31⁺ recent thymic emigrant (RTE) T_regs during normal pregnancy and in the presence of pre‐eclampsia or haemolysis elevated liver enzymes low platelet (HELLP)‐syndrome, we used six‐colour flow cytometric analysis to determine the changes in the composition of the ICOS⁺ and ICOS^? T_reg pools with CD45RA⁺CD31⁺ RTE T_regs, CD45RA⁺CD31^? mature naive (MN) T_regs, CD45RA^?CD31⁺ and CD45RA^?CD31^? memory T_regs. With the beginning of pregnancy until term, we observed a strong differentiation of both ICOS⁺ and ICOS^?CD45RA⁺CD31⁺ RTE, but not CD45RA⁺CD31^? MN T_regs, into CD45RA^?CD31^? memory T_regs. At the end of pregnancy, the onset of spontaneous term labour was associated with a significant breakdown of ICOS⁺CD45RA^?CD31^? memory T_regs. However, in the presence of pre‐eclampsia, there was a significantly increased differentiation of ICOS⁺ and ICOS^?CD45RA⁺CD31⁺ RTE T_regs into CD45RA^?CD31⁺ memory T_regs, wherein the lacking differentiation into CD45RA^?CD31^? memory T_regs was partially replaced by the increased differentiation of ICOS⁺ and ICOS^?CD45RA⁺CD31^? MN T_regs into CD45RA^?CD31^? memory T_regs. In patients with HELLP syndrome, this alternatively increased differentiation of CD45RA^?CD31^? MN T_regs seemed to be exaggerated, and presumably restored the suppressive activity of magnetically isolated ICOS⁺ and ICOS^? T_regs, which were shown to be significantly less suppressive in pre‐eclampsia patients, but not in HELLP syndrome patients. Hence, our findings propose that the regular differentiation of both ICOS⁺ and ICOS^?CD45RA⁺CD31⁺ RTE T_regs ensures a healthy pregnancy course, while their disturbed differentiation is associated with the occurrence of pre‐eclampsia and HELLP syndrome.     

Early dynamics of T helper cell cytokines and T regulatory cells in response to treatment of active Mycobacterium tuberculosis infection

Biomarkers that can identify tuberculosis (TB) disease and serve as markers for efficient therapy are requested. We have studied T cell cytokine production [interferon (IFN)-γ, interleukin (IL)-2, tumour necrosis factor (TNF)-α] and degranulation (CD107a) as well as subsets of CD4⁺ T regulatory cells (T_regs) after in-vitro Mycobacterium tuberculosis (Mtb) antigen stimulation [early secretory antigenic target (ESAT)-6, culture filtrate protein (CFP)-10, antigen 85 (Ag85)] in 32 patients with active tuberculosis (TB) disease throughout 24 weeks of effective TB treatment. A significant decline in the fraction of Mtb-specific total IFN-γ and single IFN-γ-producing T cells was already observed after 2 weeks of treatment, whereas the pool of single IL-2⁺ cells increased over time for both CD4⁺ and CD8⁺ T cells. The T_reg subsets CD25^highCD127^low, CD25^highCD147⁺⁺ and CD25^highCD127^lowCD161⁺ expanded significantly after Mtb antigen stimulation in vitro at all time-points, whereas the CD25^highCD127^lowCD39⁺ T_regs remained unchanged. The fraction of CD25^highCD127^low T_regs increased after 8 weeks of treatment. Thus, we revealed an opposing shift of T_regs and intracellular cytokine production during treatment. This may indicate that functional signatures of the CD4⁺ and CD8⁺ T cells can serve as immunological correlates of early curative host responses. Whether such signatures can be used as biomarkers in monitoring and follow-up of TB treatment needs to be explored further.     

Down syndrome, autoimmunity and T regulatory cells

Autoimmune diseases are more represented in Down syndrome (DS) individuals compared to chromosomally normal people. Natural T regulatory cells (nT_reg) have been considered to be primary in the role of controlling the intensity and targets of the immune response. We have investigated the phenotypical and functional alteration of nT_reg in a group of DS people. The phenotypical characteristic of T_reg cells of 29 DS was analysed and compared with an age‐matched healthy control group. The inhibitory potential of CD4⁺CD25^highCD127^low T regulatory cells was evaluated on autologous CD4⁺CD25^‐ T cell proliferation in response to activation with a mytogenic pan‐stimulus (anti‐CD2, anti‐CD3 and anti‐CD28 antibodies). The CD4⁺CD25^high cells in the DS and control groups were 2·692 ± 0·3808%, n = 29 and 1·246 ± 0·119, n = 29%, respectively (P = 0.0007), with a percentage of forkhead box protein 3 (FoxP3)‐expressing cells of 79·21 ± 3·376%, n = 29 and 59·75 ± 4·496%, respectively (P = 0.0015). CD4⁺CD25⁺FoxP3⁺ cells were increased in peripheral blood from DS subjects (DS mean 5·231 ± 0·6065% n = 29, control mean 3·076 ± 0·3140% n = 29). The majority of CD4⁺CD25^high were CD127^low and expressed a high percentage of FoxP3 (natural T_reg phenotype). While the proliferative capacity of DS T cells was not altered significantly compared to normal individuals, a reduced inhibitory potential of T_reg compared to healthy controls was clearly observed (mean healthy control inhibition in T_eff : T_reg 1:1 co‐culture: 58·9% ± 4·157%, n = 10 versus mean DS inhibition in T_eff : T_reg 1:1 co‐culture: 39·8 ± 4·788%, n = 10, P = 0.0075; mean healthy control inhibition in T_eff : T_reg 1:0·5 co‐culture: 45·10 ± 5·858%, n = 10 versus DS inhibition in T_eff : T_reg 1:0·5 co‐culture: 24·10 ± 5·517%, n = 10, P = 0.0177). DS people present an over‐expressed peripheral nT_reg population with a defective inhibitory activity that may partially explain the increased frequency of autoimmune disease.     

n‐Butyrate Anergized Effector CD4+ T Cells Independent of Regulatory T cell Generation or Activity

CD4⁺ T cell anergy reflects the inability of CD4⁺ T cells to respond functionally to antigenic stimulation through proliferation or IL‐2 secretion. Histone deacetylase (HDAC) inhibitors have been shown to induce anergy in antigen‐activated CD4⁺ T cells. However, questions remain if HDAC inhibitors mediate anergy through direct action upon activated CD4⁺ T cells or through the generation and/or enhancement of regulatory T (T_reg) cells. To assess if HDAC inhibitor n‐butyrate induces anergy independent of the generation or expansion of FoxP3⁺ T_reg cells in vitro, we examine n‐butyrate‐treated murine CD4⁺ T cells for anergy induction and FoxP3⁺ T_reg activity. Whereas n‐butyrate decreases CD4⁺ T cell proliferation and IL‐2 secretion, n‐butyrate did not augment FoxP3 protein production or confer a suppressive phenotype upon CD4⁺ T cells. Collectively, these data suggest that HDAC inhibitors can facilitate CD4⁺ T cell functional unresponsiveness directly and independently of T_reg cell involvement.     

Regulatory T cells and chronic immune activation in human immunodeficiency virus 1 (HIV-1)-infected children

The function of CD4⁺ T cells with regulatory activity (T_regs) is the down-regulation of immune responses. This suppressive activity may limit the magnitude of effector responses, resulting in failure to control human immunodeficiency virus 1 (HIV-1) infection, but may also suppress chronic immune activation, a characteristic feature of HIV-1 disease. We evaluated the correlation between viral load, immune activation and T_regs in HIV-1-infected children. Eighty-nine HIV-1-infected children (aged 6–14 years) were included in the study and analysed for HIV-1 plasmaviraemia, HIV-1 DNA load, CD4 and CD8 cell subsets. T_reg cells [CD4⁺ CD25^highCD127^lowforkhead box P3 (FoxP3^high)] and CD8-activated T cells (CD8⁺CD38⁺) were determined by flow cytometry. Results showed that the number of activated CD8⁺CD38⁺ T cells increased in relation to HIV-1 RNA plasmaviraemia (r = 0·403, P < 0·0001). The proportion of T_regs also correlated positively with HIV-1 plasmaviraemia (r = 0·323, P = 0·002), but correlated inversely with CD4⁺ cells (r = −0·312, P = 0·004), thus suggesting a selective expansion along with increased viraemia and CD4⁺ depletion. Interestingly, a positive correlation was found between the levels of T_regs and CD8⁺CD38⁺ T cells (r = 0·305, P = 0·005), and the percentage of T_regs tended to correlate with HIV-1 DNA load (r = 0·224, P = 0·062). Overall, these findings suggest that immune activation contributes to the expansion of T_reg cells. In turn, the suppressive activity of T_regs may impair effector responses against HIV-1, but appears to be ineffective in limiting immune activation.     

Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications

Although CD4⁺/CD25⁺ T regulatory cells (T_regs) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell‐separation strategy complying with good manufacturing practice guidelines. We isolated T_regs from standard leukapheresis products using double‐negative selection (anti‐CD8 and anti‐CD19 monoclonal antibodies) followed by positive selection (anti‐CD25 monoclonal antibody). The final cell fraction (CD4⁺/CD25⁺) showed a mean purity of 93·6% ± 1·1. Recovery efficiency was 81·52% ± 7·4. The CD4⁺/CD25^+bright cells were 28·4% ± 6·8. The CD4⁺/CD25⁺ fraction contained a mean of 51·9% ± 15·1 FoxP3 cells and a mean of 18·9% ± 11·5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4⁺CD25⁺FoxP3⁺ cells were in line with flow cytometric results. In Vβ spectratyping the complexity scores of CD4⁺/CD25⁺ cells and CD4⁺/CD25^‐ cells were not significantly different, indicating that T_regs had a broad T cell receptor repertoire. The inhibition assay showed that CD4⁺/CD25⁺ cells inhibited CD4⁺/CD25^‐ cells in a dose‐dependent manner (mean inhibition percentages: 72·4 ± 8·9 [ratio of T responder (T_resp) to T_regs, 1:2]; 60·8% ± 20·5 (ratio of T_resp to T_regs, 1:1); 25·6 ± 19·6 (ratio of T_resp to T_regs, 1:0·1)). Our study shows that negative/positive T_reg selection, performed using the CliniMACS device and reagents, enriches significantly CD4⁺CD25⁺FoxP3⁺ cells endowed with immunosuppressive capacities. The CD4⁺CD25⁺FoxP3⁺ population is a source of natural T_reg cells that are depleted of CD8⁺ and CD4⁺/CD25^‐ reacting clones which are potentially responsible for triggering graft‐versus‐host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft‐versus‐tumour effect.     

T‐bet regulates differentiation of forkhead box protein 3+ regulatory T cells in programmed cell death‐1‐deficient mice

Programmed cell death‐1 (PD‐1) plays an important role in peripheral T cell tolerance, but whether or not it affects the differentiation of helper T cell subsets remains elusive. Here we describe the importance of PD‐1 in the control of T helper type 1 (Th1) cell activation and development of forkhead box protein 3 (FoxP3⁺) regulatory T cells (T_regs). PD‐1‐deficient T cell‐specific T‐bet transgenic (P/T) mice showed growth retardation, and the majority died within 10 weeks. P/T mice showed T‐bet over‐expression, increased interferon (IFN)‐γ production by CD4⁺ T cells and significantly low FoxP3⁺ T_reg cell percentage. P/T mice developed systemic inflammation, which was probably induced by augmented Th1 response and low FoxP3⁺ T_reg count. The study identified a unique, previously undescribed role for PD‐1 in Th1 and T_reg differentiation, with potential implication in the development of Th1 cell‐targeted therapy.     

Th17, synchronically increased with Tregs and Bregs,promoted by tumour cells via cell‐contact in primary hepatic carcinoma

Documented reports about T helper type 17 (Th17) cells have revealed that Th17 plays a critical role in inflammation and autoimmunity diseases. However, the role of Th17 in cancer remains contradictory. The interplay between Th17 and tumour cells in the tumour microenvironment of primary hepatic carcinoma (PHC) needs to be explored further and the relationship between Th17, regulatory T cells (T_regs) and regulatory B cells (B_regs) has not been defined completely. In this study, numerous experiments were undertaken to elucidate the interaction of Th17 and T_reg/B_reg cells involved in PHC. Our work demonstrated that an increased Th17 was detected in the peripheral circulation and in tumour tissues in PHC patients. In addition, increases in peripheral blood Th17 corresponded with tumour–node–metastasis (TNM) stage progression. Also, further studies indicated that Th17 cells were promoted by tumour cells in the PHC tumour microenvironment through both contact‐dependent and ‐independent mechanisms, but cell‐contact played the major important role in promoting the production and proliferation of Th17. When isolated CD4⁺CD25⁺CD127^low T_regs and CD4⁺CD25^–CD127⁺ non‐T_regs were cultured with autologous tumour cells, it implied that the phenotype of Th17 and T_regs was modified by tumour cells in the tumour microenvironment. As well as this, Th17 cells were also found to correlate positively with CD4⁺forkhead box protein 3⁺ T_regs and CD19⁺CD5⁺CD1d^hi B_regs in PHC. Notably, Th17 increased synchronically with T_regs and B_regs in PHC. These findings may provide new clues to reveal the mechanisms of immune escape in PHC.     

In‐vitro effect of pembrolizumab on different T regulatory cell subsets

Programmed death‐1 (PD‐1) and interactions with PD‐ligand 1 (PD‐L1) play critical roles in the tumour evasion of immune responses through different mechanisms, including inhibition of effector T cell proliferation, reducing cytotoxic activity, induction of apoptosis in tumour‐infiltrating T cells and regulatory T cell (T_reg) expansion. Effective blockade of immune checkpoints can therefore potentially eliminate these detrimental effects. The aim of this study was to investigate the effect of anti‐PD‐1 antibody, pembrolizumab, on various T_reg subpopulations. Peripheral blood mononuclear cells (PBMC) from healthy donors (HD) and primary breast cancer patients (PBC) were treated in vitro with pembrolizumab, which effectively reduced PD‐1 expression in both cohorts. We found that PD‐1 was expressed mainly on CD4⁺CD25⁺ T cells and pembrolizumab had a greater effect on PD‐1 expression in CD4⁺CD25^? T cells, compared to CD4⁺CD25⁺ cells. In addition, pembrolizumab did not affect the expression levels of T_reg‐related markers, including cytotoxic T lymphocyte antigen‐4 (CTLA‐4), CD15s, latency‐associated peptide (LAP) and Ki‐67. Moreover, we report that CD15s is expressed mainly on forkhead box P3 (FoxP3)^?Helios⁺ T_reg in HD, but it is expressed on FoxP3⁺Helios^? T_reg subset in addition to FoxP3^?Helios⁺ T_reg in PBC. Pembrolizumab did not affect the levels of FoxP3^+/?Helios^+/? T_reg subsets in both cohorts. Taken together, our study suggests that pembrolizumab does not affect T_reg or change their phenotype or function but rather blocks signalling via the PD‐1/PD‐L1 axis in activated T cells.     

Deep sequencing of the TCR‐β repertoire of human forkhead box protein 3 (FoxP3)+ and FoxP3– T cells suggests that they are completely distinct and non‐overlapping

Maintenance of peripheral tolerance requires a balance between autoreactive conventional T cells (T_conv) and thymically derived forkhead box protein 3 (FoxP3)⁺ regulatory T cells (tT_regs). Considerable controversy exists regarding the similarities/differences in T cell receptor (TCR) repertoires expressed by T_conv and tT_regs. We generated highly purified populations of human adult and cord blood T_conv and tT_regs based on the differential expression of CD25 and CD127. The purity of the sorted populations was validated by intracellular staining for FoxP3 and Helios. We also purified an overlap group of CD4 T cells from adult donors to ensure that considerable numbers of shared clonotypes could be detected when present. We used deep sequencing of entire TCR‐β CDR3 sequences to analyse the TCR repertoire of T_conv and tT_regs. Our studies suggest that both neonatal and adult human T_conv and tT_reg cells are, in fact, entirely distinct CD4 T cell lineages.     

Association between discordant immunological response to highly active anti‐retroviral therapy,regulatory T cell percentage,immune cell activation and very low‐level viraemia in HIV‐infected patients

The mechanisms sustaining the absence of complete immune recovery in HIV‐infected patients upon long‐term effective highly active anti‐retroviral therapy (HAART) remain elusive. Immune activation, regulatory T cells (T_regs) or very low‐level viraemia (VLLV) have been alternatively suspected, but rarely investigated simultaneously. We performed a cross‐sectional study in HIV‐infected aviraemic subjects (mean duration of HAART: 12 years) to concomitantly assess parameters associated independently with inadequate immunological response. Patients were classified as complete immunological responders (cIR, n = 48) and inadequate immunological responders (iIR, n = 39), depending on the CD4⁺ T cell count (> or < 500/mm³). Clinical and virological data (including very low‐level viraemia) were collected. In parallel, immunophenotyping of CD4⁺ lymphocytes, including T_reg subsets, and CD8⁺ T cells was performed. Percentages of activated CD4⁺ T cells, T_regs, effector T_regs and terminal effector T_regs were found to be significantly elevated in iIR. Neither the percentage of activated CD8⁺ T cells nor VLLV were found to be associated with iIR. In the multivariate analysis, nadir of CD4⁺ T cell count and percentage of T_regs were the only two parameters associated independently with iIR [odds ratio (OR) = 2·339, P = 0·001, and OR = 0·803, P = 0·041]. We present here the largest study investigating simultaneously the immune response to long‐term HAART, activation of CD4⁺ and CD8⁺ T cells, T_reg percentages and very low‐level viraemia. Causative interactions between T_regs and CD4⁺ T cells should now be explored prospectively in a large patients cohort.     

Transforming growth factor beta 1 plays an important role in inducing CD4+CD25+forhead box P3+ regulatory T cells by mast cells

The role of mast cells (MCs) in the generation of adaptive immune responses especially in the transplant immune responses is far from being resolved. It is reported that mast cells are essential intermediaries in regulatory T cell (T_reg) transplant tolerance, but the mechanism has not been clarified. To investigate whether bone marrow‐derived mast cells (BMMCs) can induce T_regs by expressing transforming growth factor beta 1 (TGF‐β1) in vitro, bone marrow cells obtained from C57BL/6 (H‐2^b) mice were cultured with interleukin (IL)‐3 (10 ng/ml) and stem cell factor (SCF) (10 ng/ml) for 4 weeks. The purity of BMMCs was measured by flow cytometry. The BMMCs were then co‐cultured with C57BL/6 T cells at ratios of 1:2, 1:1 and 2:1. Anti‐CD3, anti‐CD28 and IL‐2 were administered into the co‐culture system with (experiment groups) or without (control groups) TGF‐β1 neutralizing antibody. The percentages of CD4⁺CD25⁺forkhead box P3 (FoxP3)⁺ T_regs in the co‐cultured system were analysed by flow cytometry on day 5. The T_reg percentages were significantly higher in all the experiment groups compared to the control groups. These changes were deduced by applying TGF‐β1 neutralizing antibody into the co‐culture system. Our results indicated that the CD4⁺ T cells can be induced into CD4⁺CD25⁺FoxP3⁺ T cells by BMMCs via TGF‐β1.     

Regulatory T cell frequencies are increased in preterm infants with clinical early‐onset sepsis

The predisposition of preterm neonates to invasive infection is, as yet, incompletely understood. Regulatory T cells (T_regs) are potential candidates for the ontogenetic control of immune activation and tissue damage in preterm infants. It was the aim of our study to characterize lymphocyte subsets and in particular CD4⁺CD25⁺forkhead box protein 3 (FoxP3)⁺ T_regs in peripheral blood of well‐phenotyped preterm infants (n = 117; 23 + 0 – 36 + 6 weeks of gestational age) in the first 3 days of life in comparison to term infants and adults. We demonstrated a negative correlation of T_reg frequencies and gestational age. T_regs were increased in blood samples of preterm infants compared to term infants and adults. Notably, we found an increased T_reg frequency in preterm infants with clinical early‐onset sepsis while cause of preterm delivery, e.g. chorioamnionitis, did not affect T_reg frequencies. Our data suggest that T_regs apparently play an important role in maintaining maternal‐fetal tolerance, which turns into an increased sepsis risk after preterm delivery. Functional analyses are needed in order to elucidate whether T_regs have potential as future target for diagnostics and therapeutics.